We postulate that terminal differentiation and aging in diptera, as in eukaryotic organisms, are brought about by the inactivation of specific genes and activation of others. We will test the hypothesis that one process of gene inactivation involves DNA methylation while, reciprocally, activation involves demethylation. Diptera will be used as a model system because we have observed a high level of 5-methylcytosine (as shown by binding of specific antibodies) in the DNA of transcriptionally inactive regions in salivary gland polytene chromosomes of Sciara coprophila and several Drosophila species, including D. melanogaster and D. virilis. The specific aims of this proposal are to determine the amount of 5-methylcytosine in polytene DNA and to compare it to the amount in diploid DNA, using biochemical techniques, including high pressure liquid chromatography. The possibility that some satellite DNAs are highly methylated in diploid DNA will be investigated in D. virilis, using biochemical, immunological and cytological methods. The relation between the extent of DNA methylation and the level of transcriptional activity of specific regions along polytene chromosomes during normal RNA and DNA puff development and regression and folloing puff induction by ecdysone or heat shock, will be investigated by cytological methods, including immunoperoxidase staining and autoradiography. We will attempt to modify the pattern of gene activity in polytene chromosomes by inducing demethylation, and try to induce methylation of diploid cell DNA by fusing somatic cells (neural ganglia and imaginal discs) with polytene salivary gland cells.